Bioactive surfaces made from surface-bound biomolecules may be used in a variety of bioassays, biosensors and other devices. For example, polymer-bound oligonucleotides find applications in hybridization-based diagnostics and in the discovery of new therapeutics based on molecular recognition. Prenatal diagnostics of genetic aberrations, identification of virus born diseases, detection of mutations of regulatory proteins controlling carcinogenesis, and novel hybridization-based identification techniques oriented to forensic or archaeology fields are some of the potential applications.
Bioactive surfaces may also play an essential role in areas other than medicine, pharmaceutics and biotechnology. Development of ultra-selective chemical sensors and absorbent surfaces are crucial for creating environmentally safe processes. Monitoring the quality of water is one of the major demands in this area. Biomolecular-based chemical sensors and filters for toxic chemicals and microorganisms (e.g., E. coli) will play a significant role in future technologies.
Proteins, and enzymes in particular, are one class of biomolecules commonly used to make bioactive surfaces. The advantages of using enzymes in bioassays and biosensors are related to their very high specificity (regio- and stereo-specificity) and versatility, mild reaction conditions (close to room temperatures and to pH neutral media), and to their high reaction rates. However, due to the poor recovery yields and reusability of free enzymes, much attention has been paid in the last few years to the development of efficient enzyme immobilization processes. Most biologically-active in vivo species, such as enzymes and antibodies, function in heterogeneous media. These environments are difficult to reproduce in vitro for industrial utilization. Immobilized enzyme systems are useful for experimental and theoretical research purposes for understanding the mechanisms of in vivo, bio-catalyzed reactions, and offer solutions for use in batch-type reactions, where there is poor adaptability to various technological designs and recovery of the enzymes is difficult.
The activity of enzymes (polypeptide molecules) are based on their complex three-dimensional structures containing sterically exposed, specific functionalities. The polypeptide chains are folded into one or several discrete units (domains), which represent the basic functional and three-dimensional structural entities. The cores of domains are composed of a combination of motifs which are combinations of secondary structure elements with a specific geometric arrangement. The molecular-structure-driven chain-folding mechanisms generate three-dimensional enzyme structures with protein molecules orienting their hydrophobic side chains toward the interior and exposing a hydrophilic surface. The —C(R)—CO—NH— based main chain is also organized into a secondary structure to neutralize its polar components through hydrogen bonds. These structural characteristics are extremely important and they make the enzyme molecules very sensitive to the morphological and functional characteristics of the potential immobilizing substrates. High surface-concentrations of enzyme-anchoring functionalities can result, for instance, in excessive enzyme-densities or multi-point connections which can “neutralize” the active sites or can alter the three-dimensional morphologies of the enzyme molecules through their mutual interaction and their interaction with the substrate surfaces. These are just a few of the factors which may be responsible for the significantly lower activities of immobilized-enzymes in comparison to the activities of free enzyme molecules. Rough substrate surface topographies or stereoregular surfaces (e.g., isotactic or syndiotactic polymers) might also influence, in a positive or negative way, the specific activities. Morphologically ordered surfaces might induce changes of the stereoregular shapes of protein molecules. It has also been found that enzymes can adopt more than one functional conformation other than its lowest potential energy state. E. S. Young et al., Anal. Chem. Vol. 69, 1977, pp. 4242, et seq.
A number of approaches have been proposed for immobilizing bioactive molecules, such as enzymes on inorganic substrates. One common approach is to use wet chemical techniques to functionalize a substrate surface and then to link the surface functionalities to free functional groups on a biomolecule through a linking molecule capable of forming covalent bonds to both. Unfortunately, these wet chemical techniques tend to be time-consuming, multi-step processes that involve the use of expensive and/or hazardous reagents.
Cold plasma processing has shown promise for the functionalization of inorganic substrates. Numerous experiments performed in recent years in plasma laboratories under various internal and external plasma conditions and reactor geometries clearly indicate that inert and reactive-gas discharges are effective for the surface modification (functionalization) of even the most inert materials, such as silica. The industrial applications of macromolecular plasma chemistry are rapidly developing. Large capacity reactors and continuous flow system plasma installations have been designed, developed and tested.